저자는 국립 연구 재단 원정대 (NRF -NRFF2011-01)과 경쟁 연구 프로그램 (NRF-CRP10-2012-07)의 지원을 인정하고 싶습니다.

The authors have no competing financial interests to disclose.

조직 공학 발판 빠르게 모든, 재생 유지, 또는 약물, 질병을 연구, 개발, 장기 교체 응용 프로그램에 대한 조직을 복구하는 데 필요한 물리적, 생화학 적 단서, 그리고 많은 다른 57를 제공하기 위해 진화하고있다. 간 조직 공학에서 차 인간 간세포 빠르게 한 번 몸에서 격리 대사 기능, 엔지니어링 발판을위한 훌륭한 필요성을 생성하고 간 기능을 유지하기 위해 플랫폼 개발을 잃게됩니다. <...

간은 혈액 해독, 생체 이물질의 대사 및 혈청 단백질의 생산을 포함하여 다양한 기능을 가진 매우 혈관 기관이다. 간 조직은 여러 종류의 세포, 담즙 누소관, 정현파 및 다른 biomatrix 조성물 및 다른 산소 농도의 영역으로 포함하는 복잡한 3 차원 마이크로 구조를 갖는다. 이러한 복잡한 구조를 고려할 때 시험 관내 (1) 적절한 간 모델을 만드는 것이 어려웠다. 그러나, 시험 관내 모델에서 기능 테스트 약물 독성 2 플랫폼 인간 간세포를 호스팅 간 3과 관련된 질환을 연구하는 수요가있다. 현재 간암 조직 공학 플랫폼 조나의 생화학 적 조성물을 분리, 또는 간장의 파라미터 중, 몇 개의 셀 (4) 즉, 공존 배양을 집중하여 간암의 복잡성을 단순화 한L 개의 미세 환경 5 유동 역학 6,7 및 biomatrix 8의 구성. biomatrix 구성은 지지체 물질, 세포 외 기질 (ECM) 단백질 조성물, 매트릭스 경도뿐만 아니라 지지체의 디자인 및 구조와 같은 파라미터로 나눌 수있다. 조정 하이드로 겔의 기계적 특성, 생체 활성 및 열화 율에 기능 부여, 합성 하이드로 겔, 특히 폴리 (에틸렌 글리콜) (PEG)을 사용하여 하이드로 겔 9 조직 공학 연구의 증가가 있었다. 간 관련 연구와 관련하여, 생체 적합성 하이드로 겔은 간 질환 3의 바이러스 감염 연구에 적용 하였다. 간세포 플랫폼 설계로서 수많은 연구는 생체 내 미세 환경에서 모방 필수적인 차원 환경과 셀 ECM 및 세포 - 세포 상호 작용을 제공하기 위해 하이드로 겔 (12, 13) 내에서 간세포 샌드위치 배양 10,11 세포 캡슐화를 이용했다. 하우버전, 이러한 플랫폼은 지지체 (14)를 통해 비 균일 한 특성을 선도, 제어 및 공간 조직의 높은 수준을 가지고 있지 않습니다. 반전 결정 콜로이드 (ICC)는 14 비계 먼저 2000 년대 초반에 도입 된 세포 배양을위한 매우 조직 차원의 발판이다. 발판의 독특한 구조는 콜로이드 성 결정 가변 직경의 콜로이드 입자의 정 합체를 이용하여 간단한 제조 공정에 기인 할 수있다. 간략하게, 프로세스를 요약하기 위해, 입자는 깔끔하게 배치 격자를 형성하는 열을 이용하여 어닐링된다. 고 표면적을 갖는 육방 충전 구형 캐비티 (15)의 중합 된 겔 결과, 유기 용매에 의한이 격자의 침출. 이 높은 순서 지지체 이전 (아크릴 아미드) 16-21, 폴리 (락트산 - 코 - 글리콜 산), 폴리에 한정 15,22-30, 합성 및 천연 재료 모두에서 만들어진 포함하되되었는지폴리 (에틸렌 글리콜) (31, 32), 폴리 (2- 히드 록시 에틸 메타 크릴 레이트) 21,33-35, 키토산 36-39. 비 오염 물질로 만들어진 ICC의 발판이 공동 14,23,40 내에서 세포 회전 타원체를 촉진하는 경향이있다. 여러 종류의 세포가 성공적으로 셀 (43, 44)을 확산이 구성에서 차별화 기능 연골 41, 골수 간질 세포 (42)를 포함하고, 줄기 밝혀졌다. 간세포 대해서는 연구 된 ICC 나 2 SiO3과 폴리 (아크릴 아미드)로 이루어지는 지지체 아니라 PEG로 수행되었다. 간단한 바이오 콘쥬 게이션 전략 (즉, EDC / NHS를 통해 아민 커플 링), ECM 단백질 공역 PEG 기반의 발판이 환경과 같은 생체 내에서 더를하고 간 기능을 향상시키기 위해 더 많은 세포 결합 부위를 입증 할 수있는, 제조 할 수있다. 이 원고 및 관련 비디오에서 세부 사항에게 ICC 지지체의 제조를 우리(허 7.5) 배양 간암에 최적화 된 폴리 (에틸렌 글리콜) 디 아크릴 레이트 (PEGDA), 하이드로 겔 및 폴리스티렌 미세 구조 격자를 사용. 우리는 비계 토폴로지 및 전지 성능의 관점에서 일반적으로 비 접착식 베어 PEGDA의 ICC의 발판과 콜라겐 코팅 PEGDA의 ICC 지지체의 차이점을 보여줍니다. 세포 생존 및 기능이 허 - 7.5 셀 행동을 평가하는 정 성적 및 정량적으로 측정한다.

<table><tbody><tr><td>0.2 ml PCR tube</td><td>Axygen Scientific</td><td>PCR-02D-C</td><td>Boil-proof</td></tr><tr><td>Gorilla Glue</td><td>Gorilla Glue, Inc.</td><td>Depends on vendor. This was purchased from a local store.</td><td></td></tr><tr><td>Glass slides</td><td>VWR</td><td>631-1575</td><td>Dimensions: 24&amp;times;60 mm&lt;sup&gt;2&lt;/sup&gt;</td></tr><tr><td>Polystyrene spheres</td><td>Fisher Scientific</td><td>TSS#4314A</td><td>Diameter = 140 &amp;micro;m; 3x10&lt;sup&gt;4&lt;/sup&gt; particles per milliliter and 1.4% size distribution</td></tr><tr><td>Ethanol</td><td>Merck</td><td>1.00983.1011</td><td>absolute for analysis EMSURE; Dilute to 70% with Milli-Q water</td></tr><tr><td>Ultrasonic Bath</td><td>Elma</td><td>S10H</td><td>Equiment</td></tr><tr><td>Furnace</td><td>Nabertherm</td><td>N7/H</td><td>Equipment</td></tr><tr><td>200 &amp;micro;l pipette tip</td><td>Axygen Scientific</td><td>T-210-Y-R-S</td><td></td></tr><tr><td>Rocking shaker</td><td>VWR</td><td>444-0142</td><td></td></tr><tr><td>Polyethylene Glycol (PEG)</td><td>Merck</td><td>1.09727.0100</td><td>M&lt;sub&gt;w&lt;/sub&gt;= 4 kDa; acrylation of PEG monomers and purification of the resulting precipitate produces a PEGDA macromer with M&lt;sub&gt;w&lt;/sub&gt; = 4.6&amp;nbsp;kDa</td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>392932</td><td>Equipment</td></tr><tr><td>Acrylate-Poly(Ethylene Glycol) - Succinimidyl Valerate</td><td>Laysan Bio</td><td>ACRL-PEG-SVA-3400-1g</td><td>M&lt;sub&gt;w&lt;/sub&gt; = 3.4 kDa</td></tr><tr><td>2-hydroxy-4&#039;-(2-hydroxyethoxy)-2-methylpropiophenone</td><td>Sigma Aldrich</td><td>410896</td><td></td></tr><tr><td>Vortex</td><td>VWR</td><td>58816-123</td><td>Equipment</td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5404 000.413</td><td></td></tr><tr><td>Paraffin Film</td><td>Parafilm M&amp;nbsp;</td><td>#PM996</td><td>Kept at 9&quot; with allows intensity of 10.84&amp;nbsp;mW/cm&lt;sup&gt;2&lt;/sup&gt;</td></tr><tr><td>Bluewave 200 UV spotlight</td><td>Blaze Technology&amp;nbsp;</td><td>120008, 122300</td><td></td></tr><tr><td>Tetrahydrofuran (THF)</td><td>Merck</td><td>107025</td><td></td></tr><tr><td>Orbital shaker</td><td>Heidolph</td><td>543-123120-00-5</td><td></td></tr><tr><td>Collagen Type I</td><td>Sigma Aldrich</td><td>C3867-1VL</td><td>From rat. 1x, w/o CaCl&lt;sub&gt;2&lt;/sub&gt; &amp;amp; MgCl&lt;sub&gt;2&lt;/sub&gt;; pH = 7.2</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Gibco</td><td>20012-027</td><td>16% W/V AQ. 10 x 10&amp;nbsp;ml</td></tr><tr><td>Paraformaldehyde</td><td>VWR</td><td>43368.9M</td><td>Equipment</td></tr><tr><td>Freezone 4.5 freeze drier</td><td>Labconco</td><td>7750020</td><td>Equipment</td></tr><tr><td>Sputter coater</td><td>Jeol Ltd.</td><td>JFC-1600</td><td>Equipment</td></tr><tr><td>Scanning Electron Microscope</td><td>Jeol Ltd.</td><td>JSM 5310</td><td></td></tr><tr><td>Anti-mouse primary antibodies against Collagen type I</td><td>Abcam</td><td>ab6308</td><td></td></tr><tr><td>Anti-mouse secondary antibody conjugated with Alexa Fluor 488</td><td>Life Technologies</td><td>A21121</td><td></td></tr><tr><td>Plate, Tissue Culture 24 Well, Flat Bottom (Nunclon)&amp;nbsp;</td><td>Bio-Rev PTE LTD</td><td>3820-024</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium(DMEM)&lt;br/&gt; 2.5 g/L Glucose w/ L-Gln</td><td>Lonza</td><td>12-604F</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>A15-151</td><td></td></tr><tr><td>Penicillin-Streptomycin (P/S)</td><td>Life Tchnologies</td><td>15140-122 E</td><td></td></tr><tr><td>100 mm Corning non-treated culture dishes</td><td>Sigma Aldrich</td><td>CLS430591</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Gibco</td><td>25200-056</td><td>Equipment; 37&amp;nbsp;&amp;deg;C, 5% Humidity</td></tr><tr><td>Forma Steri-Cycle CO&lt;sub&gt;2&lt;/sub&gt; Incubators</td><td>Thermofisher Scientific</td><td>371</td><td></td></tr><tr><td>Hausser Bright-Line Phase Hemacytometer</td><td>Thermofisher Scientific</td><td>02-671-6</td><td></td></tr><tr><td>Live/Dead Viability/Cytotoxicity Kit for mammalian cells</td><td>Life Technologies</td><td>L3224&amp;nbsp;</td><td></td></tr><tr><td>CCK-8 Assay</td><td>Dojindo Laboratories</td><td>CK04-11</td><td>Monosodium-salt reagent (MSR)</td></tr><tr><td>Infinite 200 PRO microplate reader&amp;nbsp;</td><td>Tecan</td><td></td><td></td></tr><tr><td>Albumin Human ELISA kit</td><td>Abcam</td><td>ab108788</td><td></td></tr><tr><td>Triton X-100</td><td>Bio-Rad</td><td>#1610407</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A2153-50G</td><td></td></tr><tr><td>Anti-mouse primary antibodies (against CYP3A4, albumin)</td><td>Santa Cruz Biotechnology</td><td>sc-53850; sc-271605</td><td></td></tr><tr><td>DAPI</td><td>Life Technologies</td><td>D3571</td><td></td></tr><tr><td>Alexa Fluor 555 labeled Phalloidin</td><td>Life Technologies</td><td>A34055</td><td></td></tr><tr><td>Trizol</td><td>Life Technologies</td><td>15596-026</td><td></td></tr><tr><td>Chloroform</td><td>VWR</td><td>22706.326</td><td></td></tr><tr><td>Isopropanol</td><td>Fisher Scientific</td><td>67-63-0</td><td></td></tr><tr><td>DPEC water</td><td>Thermofisher Scientific</td><td>AM9916</td><td></td></tr><tr><td>Nanodrop 2000c Spectrophotometer</td><td>Thermofisher Scientific</td><td>ND-2000</td><td></td></tr><tr><td>iScript Reverse Transcription Supermix&amp;nbsp;</td><td>Bio-Rad Laboratories</td><td>1708840</td><td></td></tr><tr><td>SYBR select Master Mix for CFX</td><td>Life Technology</td><td>4472937</td><td></td></tr><tr><td>Primers (to be chosen)</td><td></td><td></td><td></td></tr><tr><td>CFX96 Real-Time System, C-1000 Touch Thermal Cycler</td><td>Bio Rad Laboratories</td><td>SOFT-CFX-31-PATCH&amp;nbsp;</td><td></td></tr></tbody></table>

fabrication of inverted colloidal crystal poly(ethylene glycol) scaffold: a three-dimensional cell culture platform for liver tissue engineering

, 생체 이물질 &#39;독성 테스트 바이러스 감염 연구 및 간암을 대상으로 약물을 개발하기위한 목적으로, 시험 관내 간세포의 기능을 유지하는 능력은 세포 적당한 생화학 적 및 기계적 신호를 수신 한 플랫폼이 필요하다. 최근 간 조직 공학 시스템은 높은 수분 보유하고 세포에 필요한 기계적인 자극을 제공 할 능력이 주어 합성 또는 천연 하이드로 겔로 구성된 3 차원 지지체를 이용 하였다. 반전 콜로이드 결정 (ICC) 지지체에 대한 관심이 증가되고있다, 높은 공간 조직 동형과 이형 세포의 상호 작용뿐만 아니라, 세포 외 기질 (ECM)의 상호 작용을 가능하게하는 최근에 개발. 여기서, 우리는 폴리 (에틸렌 글리콜) 디 아크릴 레이트 (PEGDA) 및 입자 침출 방법을 이용하여 ICC 지지체를 제조하는 프로토콜을 기술한다. 간략하게, 격자는 미세 입자로부터 만들어지는 전 후 polymeR 용액 중합 적절히 첨가되고, 입자는 유기 용매 (예 : 테트라 히드로 푸란)을 사용하여 제거하거나 침출된다. 제어 된 기공 크기 및 미디어보다 쉽게​​ 세포에 도달 할 수 있도록 상호 연관성이있는 다공성 지지체의 격자 결과의 용해. 이 독특한 구조는 세포뿐만 아니라 쉽게 기공 사이의 통신, 및 단백질과 PEGDA의 ICC 지지체는 전지 성능에 현저한 효과를 나타낸다 코팅 할 수있는 능력에 부착하기위한 고 표면적을 허용한다. 우리는 가능성의 관점에서, 골격의 형태뿐만 아니라, 간암 세포 (허 7.5) 동작을 분석하고 ICC 구조 ECM 코팅의 효과를 조사하는 기능을한다. 전반적으로,이 논문은 조직 공학에서 다양한 응용 프로그램, 특히 간 조직 공학을 가진 새로운 발판의 상세한 프로토콜을 제공합니다.

국제 형사 재판소 지지체의 구조 특성 및 간세포를 배양의 각 ICC의 발판 조건의 효능의 비교에 대한 대표적인 결과가 표시하고 아래에 설명되어 있습니다. 이러한 결과에서 사용 ICC 골격 조건은 0 μg의 / ㎖ (맨발), 20 ㎍ / ㎖ (콜라겐 20), 200 ㎍ / ㎖ (콜라겐 200) 및 400 ㎍ / ㎖ (400 콜라겐) 및 초기의 콜라겐 코팅되어 허-7.5 셀 시드 수는 1 × 6입니다. <p class="jove_content...

Watch this Scientific Journal Video about Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering at JoVE.com

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

반전 콜로이드 결정체 폴리 (에틸렌 글리콜) 비계의 제작 : 간 조직 공학을위한 삼차원 세포 배양 플랫폼

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

fabrication-inverted-colloidal-crystal-poly-ethylene-glycol-scaffold

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

9.8K 조회수.  Nanyang Technological University. This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

비디오: Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

기계 다이나믹 셀 문화 Microfabricated 플랫폼

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

연구

생체공학

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

3 차원 세포 배양을위한 자기보고 발판

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

3 차원 세포 배양을위한 폐 세포 외 매트릭스에서 조정 히드로 겔

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

조직 특이 적 세포 배양 및 전달 플랫폼과 같은 세포 외 기질 유래 거품과 마이크로 담체의 제조

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

3D 공간 세포 배양을위한 세포 발판으로 생체 적합성 액정 엘라스토머 거품의 합성

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

주 사용 재생 치료와 높은 처리량 마약 검사에 대 한 3D Microtissues

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

3 차원 펩 티 드 건설 기계에 포유류 세포 배양

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Fabrication of a Crystalline Nanocellulose Embedded Agarose Biomaterial Ink for Bone Marrow-Derived Mast Cell Culture

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto which cells are deposited. Because many biomaterial inks can be potentially cytotoxic to primary cells, it is necessary to determine the biocompatibility of these hydrogel composites prior to their utilization in costly 3D tissue engineering processes. Some 3D culture methods, including bioprinting, require that cells be embedded into a 3D matrix, making it difficult to extract and analyze the cells for changes in viability and biomarker expression without eliciting mechanical damage. This protocol describes as proof of concept, a method to assess the biocompatibility of a crystalline nanocellulose (CNC) embedded agarose composite, fabricated into a 24-well culture system, with mouse bone marrow-derived mast cells (BMMCs) using flow cytometric assays for cell viability and biomarker expression.
After 18 h of exposure to the CNC/agarose/D-mannitol matrix, BMMC viability was unaltered as measured by propidium iodide (PI) permeability. However, BMMCs cultured on the CNC/agarose/D-mannitol substrate appeared to slightly increase their expression of the high-affinity IgE receptor (Fc&#949;RI) and the stem cell factor receptor (Kit; CD117), although this does not appear to be dependent on the amount of CNC in the bioink composite. The viability of BMMCs was also assessed following a time course exposure to hydrogel scaffolds that were fabricated from a commercial biomaterial ink composed of fibrillar nanocellulose (FNC) and sodium alginate using a 3D extrusion bioprinter. Over a period of 6-48 h, the FNC/alginate substrates did not adversely affect the viability of the BMMCs as determined by flow cytometry and microtiter assays (XTT and lactate dehydrogenase). This protocol describes an efficient method to rapidly screen the biochemical compatibility of candidate biomaterial inks for their utility as 3D scaffolds for post-print seeding with mast cells.

This protocol highlights a method to rapidly assess the biocompatibility of a crystalline nanocellulose (CNC)/agarose composite hydrogel biomaterial ink with mouse bone marrow-derived mast cells in terms of cell viability and phenotypic expression of the cell surface receptors, Kit (CD117) and high-affinity IgE receptor (Fc&#949;RI).

골수 유래 마스트 세포 배양을 위한 결정성 나노셀룰로오스 내장 아가로즈 생물재료 잉크의 제조

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto ...

Assembloid Technology: Pioneering Liver Development Research

In Vitro Cultivation Techniques for Modeling Liver Organogenesis, Building Assembloids, and Designing Synthetic Tissues using Human Cell Lines

Chronic liver disease has reached epidemic proportions, affecting over 800 million people globally. The current treatment, orthotopic liver transplantation, has several limitations. Promising solutions have emerged in the field of liver regenerative medicine, with liver organogenesis holding significant potential. Early liver organogenesis, occurring between E8.5 and 11.5, involves the formation of epithelial-mesenchymal interactions leading to morphogenesis, hepatic cord formation, and collective migration. However, there is a lack of methods for in vitro modeling of this process. In this study, a detailed series of methods is presented, enabling the modeling of various stages and aspects of liver organogenesis with human cell lines. In one method series, assembloid technology with hepatic (HEP) and mesenchymal (MES) spheroids are utilized, replicating early structures found in liver organogenesis, modeling early morphogenesis, and demonstrating interstitial cell migration as seen in vivo. These innovative assembloid systems help identify factors influencing assembloid formation and migration. HEP spheroid cultivation systems were also employed to model collective migration and branching morphogenesis. Mesenchymal-conditioned media (M-CM) plays a significant role in initiating dose-dependent branching migration. All presented methods were shown to be highly reproducible with a high success rate. By linking these events together, our work will enable recreating sequential, morphogenetic events to reverse engineer organogenesis in vitro and in vivo.

Organoids revolutionize personalized tissue modeling for organ development, drug discovery, and disease research. Organoid engineering advances into creating intricate synthetic tissues. The aim is to integrate morphogenesis, assembloid technology, and biomatrices to advance tissue engineering. This protocol aids in modeling liver organogenesis and establishing guidelines for synthetic tissue construction.

Chronic liver disease has reached epidemic proportions, affecting over 800 million people globally. The current treatment, orthotopic liver ...

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can progress to fibrosis and eventually to cirrhosis, a significant risk factor for hepatocellular carcinoma. The extracellular matrix (ECM) provides structural support and maintains liver homeostasis via matricellular signals. Liver fibrosis results from an imbalance in the dynamic ECM remodeling process and is characterized by excessive accumulation of structural elements and associated changes in glycosaminoglycans. The typical fibrosis pattern of NASH is called &#34;chicken wire,&#34; which usually consists of zone 3 perisinusoidal/pericellular fibrosis, based on features observed by Masson&#39;s trichrome stain and Picrosirius Red stains. However, these traditional thin two-dimensional (2D) tissue slide-based imaging techniques cannot demonstrate the detailed three-dimensional (3D) ECM structural changes, limiting the understanding of the dynamic ECM remodeling in liver fibrosis.
The current work optimized a fast and efficient protocol to image the native ECM structure in the liver via decellularization to address the above challenges. Mice were fed either with chow or fast-food diet for 14 weeks. Decellularization was performed after in situ portal vein perfusion, and the two-photon microscopy techniques were applied to image and analyze changes in the native ECM. The 3D images of the normal and NASH livers were reconstituted and analyzed. Performing in situ perfusion decellularization and analyzing the scaffold by two-photon microscopy provided a practical and reliable platform to visualize the dynamic ECM remodeling in the liver.

The present protocol optimizes the liver in situ perfusion/decellularization and two-photon microscopy methods to establish a reliable platform to visualize the dynamics of extracellular matrix (ECM) remodeling during non-alcoholic steatohepatitis (NASH).

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can ...